The present invention relates to the growth of cells in culture. More particularly, the present invention provides methods and compositions for increasing cell survival, cell proliferation and/or cell differentiation along specific pathways by growing the cells in low ambient oxygen conditions.
The sympathoadrenal (SA) lineage of the neural crest gives rise to all of the catecholaminergic derivatives of the peripheral nervous system (PNS). These include sympathetic neurons, adrenal chromaffin cells, carotid body cells, and Small Intensely Fluorescent (SIF) cells (Doupe, et al., 1985, at p. 2119 and p. 2143). The terminal differentiation and plasticity of SA derivatives have been intensively studied for over a decade (reviewed in Anderson, et al., 1993). Although multipotent neural crest progenitors have been observed to give rise to SA derivatives in vivo (Fraser, 1991), relatively little is known about how neural crest stem cells (NCSCs) become committed to this sublineage. Bone Morphogenetic Proteins (BMPs) -2, -4 and -7 have been identified as inducers of SA marker expression in mass cultures of avian and mammalian neural crest cells (Varley, et al., 1995; Varley, et al., 1996; Reissman, et al, 1996; and Lo, et al., 1999). Such BMPs can also induce autonomic neuron differentiation in clonal cultures of mammalian NCSCs (Stemple, 1992), but they do not induce SA lineage markers (Shah, et al., 1996). The inability to achieve SA lineage differentiation in clonal cultures of NCSCs has severely hampered further study of the mechanisms regulating this important lineage restriction process.
The sympathetic potential of NCSCs is of clinical as well as basic interest because sympathetic lineage cells synthesize dopamine and dopaminergic cells are used to alleviate Parkinson""s disease (reviewed in Gage, et al., 1989). Parkinson""s disease is a relatively common neurodegenerative disorder caused by the loss of dopaminergic neurons in the substantia nigra. The transplantation of a wide variety of dopaminergic cell types, including fetal mesencephalon (Freed, et al., 1992) and neural crest-derived adrenal chromaffin cells (Date, 1996) or carotid body cells (Luquin, 1999) into the substantia nigra can ameliorate the symptoms of Parkinson""s disease; however, the supply of fetal tissue is very limited and even autologous transplants of dopaminergic cells may sometimes be unsuitable (Date, 1996; Stoddard, et al., 1989). These constraints have prompted extensive efforts to identify alternate sources of dopaminergic neurons (Zawada, 1998) and to expand dopaminergic cells in culture (Studer, 1998).
Therefore, it is desirable to understand the differentiation pathways of neural crest stem cells. Moreover, it is desirable to provide methods of enhancing differentiation, proliferation and/or survival of neural crest stem cells. Furthermore, it is desirable to expand stem cell or progenitor numbers in culture and form neurons in culture, particularly dopaminergic or noradrenergic neurons in culture.
The present invention is directed to growing cells in low ambient oxygen conditions in order to mimic the physiological oxygen conditions with greater fidelity. The growth of these cells in such conditions provides certain surprising and unexpected results. These results are exploited and described in further detail herein. More particularly, the present invention describes methods that may independently be useful in increasing cell survival, cell proliferation and/or cell differentiation along specific pathways.
In one embodiment, a method of culturing at least one neural crest stem cell (NCSC) to enhance survival, proliferation or differentiation of the NCSC is provided. The method comprises culturing the NCSC(s) in low ambient oxygen conditions, wherein said survival, proliferation or differentiation is enhanced compared to a control NCSC cultured in ambient oxygen conditions.
In one aspect, a method is provided for enhancing differentiation of the NCSC wherein differentiation is to a neuron. In a preferred embodiment, differentiation is to a cell of the sympathoadrenal lineage or the cholinergic lineage. In another embodiment, differentiation is to a sensory neuron. In another aspect, differentiation is to a phenotype which produces dopamine and norepinephrine. In yet another aspect, differentiation is to adrenal chromaffin cells, small intensely fluorescent cells or sympathetic neurons.
The neural crest stem cell can be a single isolated cell. Moreover, the neural crest stem cell can be from a primary source or culture.
The methods provided can be applied in a number of ways including storing and forming cells which can be used in transplantation into subjects. Subjects can be individuals suffering from neurodegenerative diseases or disorders or animal models of the same.
In a further aspect, a method of culturing at least one undifferentiated cell to form a cell which produces dopamine and norepinephrine is provided. In one embodiment, the method comprises culturing said cell(s) in low ambient oxygen conditions to form a cell which produces dopamine and norepinephrine.
Other features and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples, while indicating preferred embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.